Fusarium wilt is a fungal infection, which is one of the most destructive diseases to affect the production of cucurbit crops. Cucurbits are members of a botanical family, which includes agricultural crops such as melon (muskmelon, honeydew and cantaloupe), watermelon, cucumbers, gourds, pumpkins, squash (summer and winter), and related plants.
Fusarium wilt is a ubiquitous disease. For example, infection with Fusarium oxysporum Schlechtend. ex Fr.f.sp. melonis Snyder & Hans. occurs throughout North America, Europe and Asia. This forma specialis of F. oxysporum infects only melons, but susceptible plants can be infected in all stages of development. The pathogen infects the roots and blocks the uptake of water and nutrients, killing the root and above-ground tissue. In infected fields, yield losses can be as high as 100% (Sherf & MacNab, "Fusarium Wilt of Muskmelon," pgs. 334-337 in VEGETABLE DISEASE AND THEIR CONTROL, 2nd ed., John Wiley & Sons, NY, 1986). Once established in field soil, the pathogen persists indefinitely. Even after prolonged periods of cultivation of non-host crops, the Fusarium wilt pathogen can still be recovered from the soil (Banihashemi & DeZeeuw, The Behavior of Fusarium Oxysporum fsp melonis in the Presence and Absence of Host Plants, Phytopathology 65:1250-1217, 1975). The only economic method for effective disease control is through introgressing the resistant gene into commercial cultivars.
Currently, four races (races 0, 1, 2, and 1,2) of the fungus are defined by their capacity to induce disease in differential melon varieties. The variety Charentais T is susceptible to all races. Doublon, which has the resistance gene Fom-1, is resistant to races 0 and 2. CM17-187, which has the resistance gene Fom-2 is resistant to races 0 and 1. Line MR-1, which has both Fom-1 and Fom-2, is resistant to races 0, 1, and 2. All, however, are susceptible to race 1,2. Race 2 is the most widely distributed in the United States and was the only race known in North America until 1985, when race 1 was discovered in the North Atlantic states (Martyn & Gordon, "Fusarium Wilt of Melon," pgs. 14-15 in Zitter et al., eds., COMPENDIUM OF CUCURBIT DISEASES, APS Press, MN, 1996). Currently, only a few Eastem-type melons are resistant to race 1 (Zuniga et al., Characterization of Pathogenic races of Fusarium Oxysporum f.sp. melonis causing Fusarium Wilt of Melon in New York, Plant Dis. 81:592-596, 1997) and commercial varieties grown in California are susceptible (Gwynn et al., A New Race of Fusarium oxysporum f.sp. melonis causing Fusarium Wilt of Muskmelon in the Central Valley of California, Plant Dis. 81:1095, 1997). Several breeding lines are highly resistant to the disease, including the widely used line MR-1 (Thomas & Jourdain, Role of Host Resistance in Management of Downy Mildew in Muskmelon, pgs. 131-135 in Proc. Clemson Univ. Cent. IPM Sypm., 1989; Thomas et al., Inheritance of Resistance to Alternana cacumerina in Cucumis melo line MR-1, Plant Dis. 74:868-870, 1990; Wang et al., A Genetic Map of Melon (Cucumis melo L.) Based on Amplified Fragment Length Polymorphism (AFLP) Markers, Theor. and Appl. Genet. 95:791-798, 1997).
Genetic control of resistance to Fusarium wilt has been studied in some detail. Two single dominate genes, Fom-1 and Fom-2, confer resistance to races 0 and 2 and races 0 and 1, respectively. However, resistance to race 1,2 appears to be polygenic (Blancard et al., IN A COLOR ATLAS OF CUCURBIT DISEASES: OBSERVATION, IDENTIFICATION AND CONTROL, John Wiley and Sons, NY, 1994; Zink & Thomas, Genetics of Resistance to Fusarium oxysporum f.sp. melonis races 0, 1, and 2 in Muskmelon Line MR-1, Phytopathology 80:1230-1232, 1990).
Traditionally, artificial inoculation techniques were used to evaluate Fusarium wilt resistance in melon breeding programs. However, this method is very time consuming. Young seedlings must be uprooted; roots pruned and dipped into an inoculum of appropriate spore concentration; seedlings replanted; and symptom development monitored constantly over four weeks or longer (Wechter et al., Development of Sequence Specific Primers which Amplify a 15 kb DNA Marker for Race 1 Fusarium Wilt Resistance in Cucumis melo, Hortscience 33:291-292, 1998; Zink & Thomas, Genetics of Resistance to Fusarium oxysporum f.sp. melonis races 0, 1, and 2 in Muskmelon Line MR-1, Phytopathology 80:1230-1232, 1990). Furthermore, using the traditional method of artificial inoculation, there are occasional escapes, even when plants are evaluated under a controlled environment; i.e., susceptible plants which survive the inoculation procedure, resulting in a mis-scoring of the phenotype.
Therefore, in view of the deficiencies of prior art methods, a rapid method which reliably identifies Fusarium resistant melon genotypes, would be highly desirable. The method of the present invention provides a reliable and rapid assay to identify race 1 Fusarium resistance in cucurbits, especially melons. This novel method uses unique DNA primers in a rapid assay using the polymerase chain reaction (PCR).
PCR is a technique by which a small fragment of deoxyribonucleic acid (DNA) can be rapidly duplicated, or cloned, to produce multiple DNA copies. The strength of the PCR technique is that it can be used to identify genetically resistant plants from minute amounts of tissue samples because it proceeds in a series of cycles, with each successive round doubling the amount of DNA present in the sample. Thus, more than one billion copies of a single DNA fragment can be made in just a few hours, by mimicking the natural DNA replication process that occurs in living cells.
There are three phases essentially in a PCR reaction. In the first phase, denaturation, the original DNA extracted from the sample is heated to a temperature of from about 90.degree. C. to 95.degree. C. for a brief period, causing the two strands of DNA to separate. In the second or annealing phase, the temperature of the sample tube is lowered over a short period of time, allowing for the added oligonucleotide primers to bind to the separated DNA strands in a complementary fashion. In the final polymerization phase the temperature of the sample mixture is again raised, to approximately 72.degree. C., allowing the polymerase enzyme to copy the segments of DNA located between annealed primer pairs rapidly. The three phases make up one complete PCR cycle, and take less than five minutes to complete.
The PCR reaction is repeated for a specified number of cycles, usually between 25 and 35, allowing the entire procedure to be completed in three to four hours. As an added advantage, this procedure can be automated with the use of commercially available thermal cyclers, allowing the entire procedure to be conducted using pre-determined parameters.
Following the completion of the PCR procedure, the samples may be run out on an electrophoresis gel to verify the presence of the desired DNA fragment. The electrophoresed products may be visualized using an ethidium bromide dye, or may be positively identified by hybridization with a probe specific for the fragments of interest.
Over the past several years PCR technology has been shown to be applicable to the diagnosis of many human, animal, and plant organisms, and a variety of clinical assays have been evaluated. Results suggest that PCR is highly sensitive and, by varying conditions used, the technique can accurately discriminate between even closely related species. However, PCR technology has never been used to identify melon cultivars having resistance to Fusarium wilt, as in the present invention, because markers linked to resistance genes have not been known. Nor have the primers and conditions suitable for identifying genetically resistant melon cultivars been known.
By identifying markers linked to Fom-2 in melon, and developing specific PCR primers from these markers, PCR technology can be used to provide an objective assay to identify melon cultivars exhibiting resistance to Fusarium wilt. Further, it is believed that the PCR assay of the present invention is applicable for identifying other cucurbit crops that exhibit Fom-2-based resistance to Fusarium wilt. Thus, the present invention allows for the rapid identification of melon plants and other cucurbits containing genotypic resistance to the race 1 Fusarium wilt pathogens and requires only small amounts of plant tissue DNA to provide a result. By providing a rapid, non-destructive assay to detect Fusarium resistant genotypes in cucurbits, the PCR assay of the present invention is useful in the marker-assisted breeding programs presently used by most seed and agricultural companies.